Ca2+ serves not only as a charge carrier to depolarize cells, but also as a broadly-targeted second messenger molecule. Hence, Ca channels are particularly important as vital links in the control of essential specialized processes like contraction and exocytosis, as well as in the modulation of universal biological activities such as gene expression and metabolism. in heart and brain, Ca channels have been implicated in the pathogenesis of ischemic disorders and long-term functional derangement. For these reasons, gaining a clear understanding of the opening and closing, or 'gating' processes of Ca channels is a quest of enormous biological consequence. Yet, major questions remain about one pervasive class of these channels, known as L-type Ca channels. The uncertainty arises in large part from Ca-sensitive inactivation, a phenomenon whereby elevated intracellular [Ca2+] speeds channel inactivation. While the existence of this physiological feedback is unmistakable, many of its rudimentary features remain unclear and present unusual technical and theoretical challenges. The overall goal of this proposal is to elucidate the kinetic, biochemical, and molecular basis of Ca-sensitive inactivation in L-type Ca channels. Single-channel and whole-cell electrophysiology will be used to investigate both native L-type Ca channels, and those expressed in mammalian cells from cDNA. Using native Ca channels, we will test and refine a new modal hypothesis of Ca-sensitive inactivation, one in which Ca2+-entry shifts channel gating from high to low open probability modes. Experiments will be performed that distinguish among channel (de)phosphorylation, calmodulin activation, and direct Ca2+ binding to the channel as the actual molecular switch for intermodal shifts. We will next undertake extensive biophysical investigation of Ca2+ currents expressed in mammalian cells from Ca channel cDNAs. Any special subunit requirements for faithful reconstitution of native Ca inactivation will be determined. Finally, the molecular basis of Ca inactivation will be examined by an iterative process that combines construction of Ca channel mutants and chimeras together with electrophysiology. Ultimately, the precise spatial dimensions of Ca channel domains that mediate Ca inactivation are to be identified. Clarification of the kinetic, biochemical, and molecular mechanisms of Ca-sensitive inactivation promises to bridge fundamental gaps in Ca channel gating. As such, this research should provide new insight into Ca channel structure-function relations, along with important constraints on the role of Ca channels in diverse biological signalling pathways.